Human BRPF1 (bromodomain PHD finger 1) is a major subunit of the histone acetyltransferase (HAT) complexes MOZ/MORF, critical in the development of the hematopoietic system and implicated in acute leukemias. BRPF1 is required for normal developmental programs and transcriptional regulation, however its role in the complex remains elusive. BRPF1 contains a cluster of multiple zinc fingers, named a PZP domain. Our recent studies demonstrate that the PZP module of BRPF1 recognizes both histone and DNA, revealing a novel link between the MOZ/MORF-mediated acetylation and the complex assembly. The molecular mechanism underlying this novel function of BRPF1 is unclear and will be elucidated in the proposed studies. We hypothesize that concomitant binding of the PZP module of BRPF1 to histone H3 tail and DNA recruits and/or stabilize the MOZ/MORF HAT complexes at chromatin, and that posttranslational modifications (PTMs) of histone H3 modulate binding of PZP and fine-tune the HAT activity. We seek to elucidate the molecular basis and functional significance of interactions of BRPF1 PZP with chromatin. This study is of fundamental importance for understanding the epigenetic mechanisms of HAT-stimulated transcriptional activation. The specific aims of this project are: (1) to elucidate the functional and structural relationship between zinc fingers of BRPF1, and (2) to determine the molecular basis and functional significance of the PZP assembly at chromatin. To define the molecular mechanism of chromatin targeting, the atomic-resolution structures of the PZP module in complex with histone peptide and DNA will be determined using NMR spectroscopy or X-ray crystallography. Specificities for PTMs of histones and the DNA sequence, and the assembly of PZP on nucleosomes will be characterized by peptide library screening, electrophoretic mobility shift, NMR and Forster Resonance Energy Transfer. The histone- and DNA-binding site residues will be mutated and the mutant proteins will be tested in vitro and in vivo to determine the role of PZP in chromatin association, BRPF1-dependent transcriptional activation, and regulation of histone acetylation and in vivo localization of the MOZ/MORF complexes. We will utilize chromatin immunoprecipitation, PCR, fluorescence microscopy and HAT assays in this context to assess how the PZP region of BRPF1 contributes to the functions of MOZ/MORF HATs. These studies will shed light on the role of BRPF1 PZP in functioning of the MOZ/MORF complexes, allowing us to build a model of signaling by BRPF1/MOZ/MORF, and will lead to a better understanding of the epigenetic mechanisms for the regulation of gene transcription and chromatin remodeling.